Cryo-Electron Microscopy Screening Automation Across Multiple Grids Using Smart Leginon

At The Simons Electron Microscopy Center, we aim to provide superb electron microscopy services, expertise, aNd training to local and national researchers and to develop innovative methods to optimize workflows. Our scope covers service, development, machine learning, and biomedical research. We are trying to answer, how can we better optimize our services?

Machine learning is changing the field of electromicroscopy. Data collection is becoming automated. Data analysis tools are rapidly becoming more efficient.

This includes denoising images, particle picking, 3G flexibility analysis, and model building. We regularly update software to account for different situations, such as different grid holes, tilted collection, or bugs identified through beta testing software in our production environment. We also integrate feedback from our users, so we are continuously providing features that are useful for them.

Smart Leginon addresses the labor-intensive and time-consuming tasks of screening one or more grids on a cryo-TEM. It enables operators and researchers to more quickly screen and analyze grids, while freeing up time to optimize grid and sample conditions in parallel. This also reduces microscope time and cost.

Smart Leginon Autoscreen offers fully unattended multigrid screening, which takes about 10 minutes to set up screening for 12 grids. Compared to manual screening, our protocol saves several hours per day. Since Smart Leginon's introduction, other free and commercial software has been developed for similar automation.

To begin, set up the Leginon interface, then navigate to Application and click Run. In the Run Application window, choose the Ptolemy application Set main to the Leginon computer and scope and camera to their respective computers. Now import presets in the Preset Manager.

Within the Square Targeting Node Settings, ensure that both Sort targets by shortest path and Enable auto targeting are selected. In the Square Node Settings, confirm that the Wait for node to process the image is checked. Add the Square preset to the list if it's not already included.

Under Advanced Settings, check Set these apertures while imaging and ensure the aperture values are correct. To adjust Hole Targeting Node Settings, check Allow for user verification of selected targets. Uncheck both Queue up targets and Skip automated hole finder.

In the Hole Node Setting, check Wait for a node to process the image and the Hole preset is in the list on the right In Advanced Settings, check Set these apertures while imaging and confirm the aperture values. In the Exposure Targeting Node Settings, select Allow for user verification of selected targets. Ensure the Queue up targets and Skip automated hole finder are unchecked.

To configure the Exposure Node Settings, ensure the Wait for a node to process the image is unchecked. Confirm that the exposure preset is included in the list on the right. In Advanced Settings, select Set these apertures while imaging, and verify that the aperture values are accurate.

In the Focus Node Settings, ensure Wait for a node to process the image is unchecked. Verify that the auto-focusing preset is listed on the right, and set the Desired autofocus accuracy to 4 x 10 to the 6 meters. In the Focus Node Focus Sequence, activate only two-beam tilt auto focusing steps.

To adjust the Z Focus Node Settings, ensure that Wait for a node to process the image is unchecked. Confirm the Hole preset is listed on the right, and set the Desired autofocus accuracy to 5 x 10 to the 5 meters. In the Z Focus Node Focus Sequence, enable only two low-magnification stage tilt steps.

After determining the grid Z height and collecting an atlas, use the Square Targeting Node in Ptolemy to locate squares. Observe the blue circles on each square. Hover over each blob to view their size as computed by Ptolemy and record the sizes of the largest and smallest blobs.

To modify the threshold settings, adjust the minimum and maximum filter range to include desirable squares and exclude undesirable ones. Press the Fine Squares button on the top tool bar. Fine-tune the filter range until the Fine Squares feature effectively targets squares.

In the Acquisition Settings, select appropriate values for the maximum number of targets and number of target group to sample. Once the parameters are satisfactory, click the Play button to proceed. Then utilize the Ruler tool in the Hole Targeting Node to measure the diameter of a hole.

In the Template Settings, ensure the measured diameter into Final Template Diameter and click Test. Adjust the diameter value until all holes display bright white peaks at their centers. Click Test in the Threshold Settings.

Modify the A value until the binarized image distinctly shows white areas solely where holes are present. In the Blobs Settings, measure a minimum distance from the edge with the ruler and input that value to exclude border targets. Filter blobs based on size, roundness, and desired quantity.

Hover over blobs to view their values, and click Test to review the current settings. Enter the radius of holes and the spacing between them in the Lattice Settings. Then click the 42 button to gauge the reference intensity value of a vacuum area.

In the Acquisition Settings, check use Subset of the acquisition targets and set the Sample Maximal value to a low number, such as 2. Define a broad range of ice thickness means and standard deviations. Use test targeting to randomize target selection based on the above values.

Click the Play button to proceed once all settings are satisfactory. In the Hole Settings, set the Shell Script to the hl.finding. sh script path in the Ptolemy installation.

Set the Minimum Sore to less than zero. Input the radius of the holes. Then click the Show Value button to measure the reference intensity value of a vacuum area.

Click Test to find the lattice of holes. In Acquisition Settings, check Use subset of the acquisition targets, and set the sample Maximal Value to a small number, such as 4. Set a wide range of ice thickness means and standard deviations.

Click the Play button when satisfied with all settings. Leginon will perform eucentric focus and collect high-magnification images, which can be seen in the Exposure Node. Check the next exposure targeting image to see if the settings are sufficient.

Once satisfied, uncheck Allow for user verification of selected targets in the Exposure Targeting and Hole Targeting Settings. Once the grid is done screening, click File, followed by Exit to close Leginon. To set up Smart Leginon Autoscreen, execute smartlegenionautoscreen.

py in a terminal window Select GY, and input a comma separated list of grid slots to the screen. Then input full for the workflow. Input the template session name for new sessions and the template sessions Z-height value.

A GY will open to input the session name and select project associations for each grid. Leave the microscope unattended or monitor it in Leginon, Appion, and the microscope computer.